Sudhakar A, Ramachandran A, Ghosh S, Hasnain S E, Kaufman R J, Ramaiah K V
Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, Andhra Pradesh, India.
Biochemistry. 2000 Oct 24;39(42):12929-38. doi: 10.1021/bi0008682.
Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.
真核起始因子2（eIF2α）α亚基上丝氨酸51残基的磷酸化抑制了eIF2B的鸟嘌呤核苷酸交换（GNE）活性，推测是通过与eIF2B形成紧密复合物来实现的。eIF2B的GNE活性受到抑制会导致eIF2循环利用和蛋白质合成受损。我们已经在杆状病毒系统中部分纯化了野生型（wt）eIF2α以及丝氨酸51残基被丙氨酸取代（51A突变体）或天冬氨酸取代（51D突变体）的eIF2α突变体。对这些突变体的分析为丝氨酸51在eIF2与eIF2B相互作用中的作用提供了新的见解。两种突变体在体外均未被磷酸化。由于双链RNA依赖性蛋白激酶（PKR）的激活，两种突变体均降低了在血红素和聚肌苷酸（poly(IC)）处理的网织红细胞裂解物中发生的eIF2α磷酸化。然而，添加51D突变体eIF2α蛋白（而非51A突变体）会促进在补充了血红素的兔网织红细胞裂解物中eIF2B的GNE活性受到抑制，在这种裂解物中内源性eIF2α磷酸化相对较少或未发生。51D突变体增强了在PKR活跃的血红素和poly(IC)处理的网织红细胞裂解物中发生的eIF2B的GNE活性抑制。我们的结果表明，由于eIF2α磷酸化增加而在网织红细胞裂解物中发生的eIF2与eIF2B蛋白之间相互作用的增强，会因添加51A突变体蛋白而显著降低，但添加51D突变体蛋白则不会。与51D突变体蛋白表现得像磷酸化的eIF2α这一观点一致，添加这种部分纯化的重组亚基（而非51A或wt eIF2α）会增加在积极翻译的补充了血红素的裂解物中eIF2与2B蛋白之间的相互作用。这些发现支持了eIF2α中丝氨酸51残基的磷酸化促进eIF2α(P)与eIF2B之间复合物形成从而抑制eIF2B的GNE活性这一观点。
